Osteoblastic differentiation and bone formation is a well orchestrated cascade of events that is necessary for normal bone development and repair/fracture healing. The objective of this application is to characterize the role of Connective Tissue Growth Factor (CTGF) on osteoblastic differentiation and bone formation, with the ultimate goal of using BMP-9 and its downstream targets (e.g. CTGF), as bone regeneration agents in patients. CTGF is a secreted protein which has four distinct modules (domains) that share structural homology to several extracellular matrix proteins. Each module may have one or more distinct functions. We have found that CTGF expression is consistently upregulated early in the pathway of Bone Morphogenetic Protein (BMP)-induced osteoblastic differentiation. Several BMPs, especially BMP9, have been shown to be potent stimulators of osteoblastic differentiation and bone formation. Interestingly, temporal regulation of CTGF expression may be essential for BMP-induced osteoblastic differentiation. In order to further understand the molecular events regulating osteogenic lineage-specific differentiation of mesenchymal stem cell and bone formation, we propose a series of hypothesis-driven experiments that will determine the role of CTGF in this well-orchestrated process. Specifically, we will determine: (1) whether specific modules of CTGF and temporal expression of CTGF regulate BMP9-induced osteoblastic differentiation in vitro;(2) if CTGF regulates BMP9-induced bone formation in a heterotopic bone formation animal model;and (3) whether temporally controlled CTGF silencing by RNA interference (RNAi)-mediated knockdown modulates BMP9-induced osteoblastic differentiation in vitro, and heterotopic bone formation in vivo. Knowledge gained from these sets of experiments will expand our understanding of the role of BMPs and CTGF on osteogenic differentiation of mesenchymal stem cells and bone formation. Both BMPs and their downstream targets, like CTGF, can have a broad range of clinical applications in a number of orthopaedic conditions, such as enhancing recalcitrant fracture healing or promoting bone regeneration in patients. LAY STATEMENT: In normal development and bone healing, there is a well-orchestrated process of stimulating precursor cells to become bone-forming cells, and the eventual formation of bone. This proposal examines the function of an important factor (Connective Tissue Growth Factor) in this complex process of bone formation, with the goal of its application in fracture healing and bone regeneration in patients.